In situ detection of methylated DNA by histo endonuclease-linked detection of methylated DNA sites: a new principle of analysis of DNA methylation.

For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. Although previous studies described methylation of isolated DNA extracted from cells and tissues using a combination of appropriate restriction endonucleases, no application to tissue cell level has been reported. Here, we report a new method, named histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequences in a tissue section. In this study, we examined changes in the methylation level of CCGG sites during spermatogenesis in paraffin-embedded sections of mouse testis. In principle, the 3\u27-OH ends of DNA strand breaks in a section were firstly labeled with a mixture of dideoxynucleotides by terminal deoxynucleotidyl transferase (TdT), not to be further elongated by TdT. Then the section was digested with Hpa II, resulting in cutting the center portion of non-methylated CCGG. The cutting sites were labeled with biotin-16-dUTP by TdT. Next, the section was treated with Msp I, which can cut the CCGG sequence irrespective of the presence or absence of methylation of the second cytosine, and the cutting sites were labeled with digoxigenin-11-dUTP by TdT. Finally, both biotin and digoxigenin were visualized by enzyme- or fluorescence-immunohistochemistry. Using this method, we found hypermethylation of CCGG sites in most of the germ cells although non-methylated CCGG were colocalized in elongated spermatids. Interestingly, some TUNEL-positive germ cells, which are frequent in mammalian spermatogenesis, became markedly Hpa II-reactive, indicating that the CCGG sites may be demethylated during apoptosis

Albumin-conjugated PEG liposome enhances tumor distribution of liposomal doxorubicin in rats

To evaluate the effect of coupling of recombinant human serum albumin (rHSA) onto the surface of poly(ethylene glycol)-modified liposorne (PEG liposome) on the in vivo disposition characteristics of liposomal doxorubicin (DXR), the pharmacokinetics and tissue distribution of DXR were evaluated after intravenous administration of rHSA-modified PEG (rHSA/PEG) liposomal DXR into tumor-bearing rats. rHSA/PEG liposome prepared using a hetero-bifunctional cross-linker, N- succinimidyl 3-(2-pyridyldithio) propionate (SPDP), efficiently encapsulated DXR (over 95%). rHSA/PEG liposomal DXR showed longer blood-circulating property than PEG liposornal DXR and the hepatic and splenic clearances of rHSA/PEG liposornal DXR were significantly smaller than those of PEG liposomal DXR. It was also demonstrated that the disposition of DXR to the heart, one of the organs for DXR-related side-effects, was significantly smaller than free DXR. Furthermore, the tumor accumulation of rHSA/PEG liposomal DXR was significantly larger than that of PEG liposomal DXR. The &#34;therapeutic index&#34;, a criterion for therapeutic outcome, for rHSA/PEG fiposornal DXR was significantly higher than PEG liposomal DXR. These results clearly indicate that rHSA-conjugation onto the surface of PEG liposome would be a useful approach to increase the effectiveness and safety of PEG liposomal DXR.</p

Inactivation of ESBL-E. coli

The prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is increasing rapidly and spreading worldwide, particularly in Asia, compared to other regions. In the last ten years, in our hospital, in particular, there has been a < 30% increase. To prevent the spread of ESBL in hospitals and the community, the ultraviolet (UV) A-light-emitting diode (LED) irradiation device was used to inactivate ESBL-E. coli in human livestock and the environment. ESBL-E. coli and E. coli bacterial samples were collected from patients at Tokushima University Hospital (Tokushima City, Japan). The UVA-LED irradiation system had 365 nm single wavelength, and the current of the circuit was set to 0.23 or 0.50 A consistently. Results demonstrated that UVA-LED was useful for the inactivation of ESBL-E. coli and E. coli. The minimum energy dosage required to inactivate ESBL-E. coli and E. coli was 40.76 J/cm2 (45 min) in the first type of UVA-LED and 38.85 J/cm2 (5 min) in the second type. There were no significant differences between ESBL-E. coli and E. coli. The inactivation of ESBL-E. coli was dependent on energy. These findings suggest that UVA-LED with 365 nm single wavelength could be useful for surface decontamination in healthcare facilities

Role of CFTR in Campylobacter jejuni infection

Campylobacter jejuni (C. jejuni) is gastroenteritis inducible food-born pathogen. Invasion and adhesion process are essential for leading gastroenteritis in C. jejuni infection process. As against bacterial strategy for efficacy invasion and adhesion, mucosal layer play a key role in defense systems, which modulated by several ion channels and transporters mediated water flux on the intestine. Cystic fibrosis transmembrane conductance regulator (CFTR) play the main role in waterfulux in intestine, and it closely related with bacterial clearance. We previously reported that C. jejuni infection suppresses CFTR channel activity in intestinal epithelial cells, however the mechanism and importance of this suppression is unclear. This study seeks to elucidate the role of CFTR in C. jejuni-infection. Using HEK293 cells that stably express wild type and mutated CFTR, we found that CFTR attenuated C. jejuni invasion, it was not involved bacterial adhesion or intracellular survival but associated with microtubule-dependent cellular transport. Moreover we revealed that CFTR attenuated function of microtubule motor protein but not microtubule stability, which causes inhibition of C. jejuni-invasion. Meanwhile, the CFTR mutant G551D-CFTR, which has defects in channel activity, suppressed C. jejuni-invasion, whereasΔF508-CFTR, which has defects in maturation, did not suppress, suggesting that CFTR suppression of C. jejuni-invasion is related to CFTR maturation but not channel activity. Taken together, mature CFTR inhibited C. jejuni invasion by regulating microtubule-mediated pathways. We suggest that CFTR plays a critical role in cellular defenses against C. jejuni-invasion, and CFTR suppression may be an initial step in promoting cellular invasion during C. jejuni-infection

What Impact does Physical Education have on Classroom Management?: Focus on a case of a novice teacher in a primary school

This article aims to address the relationship between physical education and classroom managemen of a novice teacher in a primary school. In conclusion, the things of this case study were as follows: (1) As a feature of the novice teacher A, the scores in "Attitude" dimension of physical education class evaluation was high, then learning discipline was retained. However, the scores in "Human Relationship" dimension of class consciousness showed low value, and it turned out that the novice teacher A was suffering from constructing good human relations within the class. (2) From the result of correlation between physical education class evaluation and classroom manegement, there was no strong relationship between physical education and classroom management of the novice teacher A. As a factor, from the interview of the novice teacher A, it was suggested that the image of classroom manegement the novice teacher A intended was not clear

New Naphthoquinone Derivatives from Fusarium Napiforme of a Mangrove Plant

Two new naphthoquinone derivatives, 6-hydroxy-astropaquinone B (1) and astropaquinone D (2) as well as the known compound 3-O-methyl-9-O-methylfusarubin (3) were isolated from Fusarium napiforme, an endophytic fungus isolated from the mangrove plant, Rhizophora mucronata. The structures of 1 and 2 were determined by 1D and 2D NMR spectroscopic analyses. Compounds 1, 2 and 3 exhibited moderate antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, 1, 2 and 3 were phytotoxic action in lettuce seeding at a concentration of 30 lg mL1

Christopher Simpson The Division-Viol, or The Art of PLAYING Ex tempore upon a GROUND. EDITIO SECVNDA Part III "The Method of ordering Division to a Ground" (3)

本訳稿はChristopher Simpson (1605頃－1669) 著 The Division-Viol, or, The Art of PLAYING Ex tempore upon a GROUND. DIVIDED INTO THREE PARTS. EDITIO SECVNDA, London, 1665 のPart III "The Method of ordering Division to a Ground" より§13～§16(pp.57-61)の全訳である